High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice

Affiliations
Department of Biomedical Sciences, Chonnam
National University Medical School, Gwangju, Republic of Korea,
Research Institute of Medical Sciences,
Chonnam National University Medical School, Gwangju, Republic of
Korea

Figures

Abstract

When expression of more than one gene is required in cells, bicistronic or
multicistronic expression vectors have been used. Among various strategies
employed to construct bicistronic or multicistronic vectors, an internal
ribosomal entry site (IRES) has been widely used. Due to the large size and
difference in expression levels between genes before and after IRES, however, a
new strategy was required to replace IRES. A self-cleaving 2A peptide could be a
good candidate to replace IRES because of its small size and high cleavage
efficiency between genes upstream and downstream of the 2A peptide. Despite the
advantages of the 2A peptides, its use is not widespread because (i) there are
no publicly available cloning vectors harboring a 2A peptide gene and (ii)
comprehensive comparison of cleavage efficiency among various 2A peptides
reported to date has not been performed in different contexts. Here, we
generated four expression plasmids each harboring different 2A peptides derived
from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea
asigna virus and porcine teschovirus-1, respectively, and evaluated
their cleavage efficiency in three commonly used human cell lines, zebrafish
embryos and adult mice. Western blotting and confocal microscopic analyses
revealed that among the four 2As, the one derived from porcine teschovirus-1
(P2A) has the highest cleavage efficiency in all the contexts examined. We
anticipate that the 2A-harboring cloning vectors we generated and the highest
efficiency of the P2A peptide we demonstrated would help biomedical researchers
easily adopt the 2A technology when bicistronic or multicistronic expression is
required.

Funding: This work was supported by the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2009-0071597 and 2009-0070204), a grant of the Korean
Health Technology R&D Project, Ministry for Health, Welfare & Family
Affairs, Republic of Korea (A100402) and the Korea Science and Engineering
Foundation through the Medical Research Center for Gene Regulation
(R13-2002-013-06002-0) at Chonnam National University. The funders had no role
in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

Competing interests: SRL is an employee of Kim & Jung Co. Ltd. All authors confirm adherence to
all the PLoS ONE policies on sharing data and materials.

Introduction

In biomedical research, the simultaneous expression of more than one gene in cells or
organisms using a single plasmid is sometimes required. To this end, several
strategies have been employed: (i) multiple promoters fused to the genes' open
reading frames (ORFs); (ii) insertion of splicing signals between genes; fusion of
genes whose expressions are driven by a single promoter; (iii) insertion of
proteolytic cleavage sites between genes; and (iv) insertion of internal ribosomal
entry sites (IRESs) between genes [1], [2].

Of these strategies, IRES has been widely used due to the following advantages: (i)
ensured coexpression of genes before and after the IRES; (ii) feasibility of adding
subcellular localization sequences to the gene after IRES; and (iii) availability of
commercial expression plasmids harboring IRES [2]. However, IRES has two major
limitations [2],
[3]. First,
the size of IRES is usually longer than 500 nucleotides, which could be a problem
when a large insert is cloned into IRES-containing viral vectors with limited
cloning capacity or when multiple IRESs are used to generate multicistronic
plasmids. Second, translation efficiency of a gene placed after the IRES is much
lower than that of a gene located before IRES.

These limitations can be overcome by a 2A peptide, a “self-cleaving”
small peptide first identified by Ryan and colleagues in the foot-and-mouth disease
virus (FMDV), a member of the picornavirus [4]. The average length of 2A peptides
is 18–22 amino acids. The designation “2A” refers to a specific
region of picornavirus polyproteins and arose from a systematic nomenclature adopted
by researchers. In FMDV, it is delineated at its own C-terminus by
‘cleavage’ (to be more precise, by ribosome skipping) and at its
N-terminus by a proteolytic cleavage or ‘trimming’ from the upstream
capsid protein 1D by the 3C/3CD proteinase. Initially, it was speculated that either
a virus-encoded proteinase or host cell proteinase might be responsible for the
cleavage [4]. Recent
reports demonstrated, however, that ribosomes skip the synthesis of the
glycyl-prolyl peptide bond at the C-terminus of a 2A peptide, leading to the
cleavage between a 2A peptide and its immediate downstream peptide [5], [6], [7]. As a result,
the cleaved-off downstream peptide has proline at its N-terminus (Fig. 1A). The term CHYSEL
(cis-acting
hydrolase
element) was coined by Pablode Felipe to reflect the
characteristics of this type of picornavirus 2A peptides [8].

2A-mediated cleavage is a universal phenomenon in all eukaryotic cells. However, it
has not been observed in prokaryotic cells [9]. In addition to the FMDV 2A
peptide, several 2A peptides have since been identified in picornaviruses, insect
viruses and type C rotaviruses [2]. Of the 2A peptides identified to date, four have been
widely used in biomedical research: FMDV 2A (abbreviated herein as F2A); equine
rhinitis A virus (ERAV) 2A (E2A); porcine teschovirus-1 2A (P2A) and
Thoseaasigna virus 2A (T2A). The former three viruses belong to
picornaviruses and the latter is an insect virus [3].

Two advantages of the 2A peptide over IRES are its short length and stoichiometric
expression of multiple proteins flanking the 2A peptide [3]. Despite these advantages, the
use of 2A peptides has not been as common in biomedical research as it should be; we
speculated that there might be two reasons. First, it is not certain which 2A
peptide has the highest cleavage efficiency. Donnelly and colleagues showed in 2001
that T2A has the highest cleavage efficiency (close to 100%) followed by E2A,
P2A and F2A [10]. In addition, Szymczak and colleagues demonstrated in 2004
that F2A and T2A have higher efficiency (close to 100%) than E2A [11]. However,
since their claims were based on in vitro transcription/translation
experiments, it remains unclear as to how the 2A peptides would act in an in
vivo context. Second, to the best of our knowledge, there is no
publicly or commercially available cloning vector harboring a ubiquitous promoter
and a 2A gene flanked by multiple cloning sites, which could make it difficult for
scientists to adopt 2A technology for coexpression of more than one gene.

To circumvent these two obstacles, we first set out to compare the cleavage
efficiency of F2A, E2A, P2A and T2A peptides in three human cell lines, zebrafish
and mice using Western blotting and confocal microscopy analyses. Second, we
embarked on constructing cloning vectors bearing a simian cytomegalovirus (sCMV)
promoter fused to a 2A gene flanked by multiple cloning sites.

Materials and Methods

Ethics statement

Animal studies were approved by the Chonnam National University Medical School
Institutional Animal Care and Use Committee (Project number:
CNUIACUC-H-2010-20).

Because anti-DsRed antibody has been successfully used to decorate mCherry
protein [12],
anti-DsRed antibody was used to visualize the mCherry protein.

Synthesis of RNA for microinjection into zebrafish embryos

Plasmids encoding NLS-EGFP-2A-mCherry-CAAX were linearized with ClaI and
in vitro transcribed with SP6 RNA polymerase using an
mMessage mMachine kit (Ambion). The resulting RNA (200 pg) was microinjected
into one cell stage embryos.

Generation of recombinant adenovirus

Recombinant adenovirus was generated as described previously [13]. In brief, a
DNA fragment encoding NLS-EGFP-2A-mCherry-CAAX was cloned into the NotI/SalI
sites of a pShuttle vector (Agilent Technologies, CA, USA). The resulting
construct was linearized with PmeI and then introduced via electroporation into
BJ5183 electroporation-competent cells harboring pAdEasy-1 (Agilent
Technologies). The resulting recombinant clones were selected by kanamycin and
digestion with restriction endonucleases. Subsequently, the verified clone was
amplified, linearized with PacI and then transfected into HEK293 A packaging
cells. The recombinant adenovirus was released from the cells through four
freeze-thaw-vortex cycles 14–20 day post-transfection, amplified through
infection of HEK293 A packaging cells and purified by CsCl ultracentrifugation.
The resulting high-titer (tissue-culture infected dose at 50%
[TCID50]) recombinant adenovirus was injected into mice
via the tail vein.

Image analysis

The images taken were assembled using Adobe Photoshop and band intensity on the
Western blots was analyzed using ImageJ.

Results

To generate versatile cloning vectors harboring genes encoding viral 2A peptides, we
inserted four different 2A sequences (P2A, T2A, E2A and F2A; Fig. 1B) [11] into a pCS4+ plasmid,
which has a simian cytomegalovirus (sCMV) promoter, SP6 and T7 RNA polymerase
binding sites, multiple cloning sites (MCSs) and an SV40 polyadenylation site [14]. The sCMV promoter
is a ubiquitous promoter and can function in Xenopus, zebrafish,
mouse and human [15]. There sulting vectors have 2A sequences flanked by several
unique recognition sites for restriction endonucleases (Fig. 1C). Nucleotide sequences encoding
Gly-Ser-Gly were added to the 5′end of the 2A sequences to improve cleavage
efficiency [11].
Sequences of the MCSs and recognition sites of unique restriction endonucleases
therein are shown in Fig.
1D.

To test whether the four 2A sequences used act properly, we fused a nuclear
localization sequence (NLS) to the N-terminus of EGFP (termed NLS-EGFP) and a
membrane localization sequence (CAAX, where X = any amino acid)
to the C-terminus of mCherry (termed mCherry-CAAX) and then inserted the resulting
NLS-EGFP and mCherry-CAAX [16] into the four different 2A cloning vectors generated. The
resulting constructs were named pNLS-EGFP-2A-mCherry-CAAX. Subsequently, we
transfected three widely used human cells lines (HEK293T [an embryonic kidney
cell line], HT1080 [a fibrosarcoma cell line] and HeLa [a
cervical cancer cell line]) with the pNLS-EGFP-2A-mCherry-CAAX and analyzed the
cleavage efficiency of the respective 2A peptides using WB and confocal microscopy
24-hr post-transfection. WB analysis revealed that all four 2As function with
various cleavage efficiency in the HEK293T (Figs. 2A and 2B), HT1080 (Figs. 3A and 3B) and HeLa cell lines (Figs. 4A and 4B), with P2A to be
the most efficient, followed by T2A, E2A and F2A. The two major byproducts indicated
by asterisks in Figure 2A were
noted as well in a previous report [17]. Of note, the byproducts did not appear in the lysate of
cells transfected with pEGFP-N1 plasmid that does not have 2A sequences (marked GFP
in Fig. S1),
raising the possibility that their production may be related to 2A mediated
‘cleavage’ activity.

HEK293T cells were transfected with the indicated plasmids. (A) WB analysis
of cleavage efficiency of the 2As in HEK293T cells. The transfected cells
were processed for WB 24 hr post-transfection. The cleavage efficiency was
assessed using GFP and DsRed antibodies to decorate NLS-EGFP and
mCherry-CAAX, respectively. Asterisks indicate two major byproducts.
Anti-β actin antibody was used as a loading control. (B) Quantitation of
cleavage efficiency of indicated 2As. Cleavage
efficiency = cleaved form/(cleaved form+uncleaved
form). The amount of each form was estimated from its band intensity on the
Western blot measured by ImageJ software. The p value was
determined by the two-tailed Student's t-test
(n = 3). (C) Confocal microscopy of the transfected
cells. Green signals in the nucleus that do not overlap with red signals
indicate cleaved NLS-EGFP. Conversely, red signals in the plasma membrane
that do not overlap with green signals represent cleaved mCherry-CAAX. In
the overlay images, yellow signals derived from overlapping of green and red
signals denote uncleaved NLS-EGFP-2A-mCherry-CAAX. The scale bar represents
20 µm.

In addition, the difference in cleavage efficiency among the 2As was confirmed by
confocal microscopy: targeting of NLS-EGFP and mCherry-CAAX produced by 2A-mediated
cleavage to the nucleus and plasma membrane, respectively, in the cell lines was the
most efficient in P2A, followed by T2A, E2A and F2A. (Figs. 2C, 3C and 4C for HEK293T, HT1080 and HeLa cells,
respectively).

To determine if the results gained from experiments with the human cultured cell
lines are recapitulated in zebrafish embryos, we generated mRNAs from
pNLS-EGFP-2A-mCherry-CAAX using SP6 RNA polymerase and microinjected the resulting
mRNAs into one-cell stage embryos. The embryos at 24 hpf were then harvested and
analyzed using WB and confocal microscopy. WB analysis exhibited that P2A has the
highest cleavage efficiency in zebrafish as well, followed by T2A, E2A and F2A
(Fig. 5A). Moreover,
confocal microscopic analysis of zebrafish embryonic retina confirmed the WB result,
as judged by the targeting of NLS-EGFP and mCherry produced by 2A-mediated cleavage
to their respective destination, just as shown for human cultured cell lines (Fig. 5B). Taken together, findings
of the experiments with zebrafish embryos recapitulated those of human cultured cell
line experiments.

One- or two-cell stage embryos were injected with in vitro
transcribed RNAs encoding the indicated proteins. (A) WB analysis revealing
cleavage efficiency in the 2As in zebrafish embryos. The injected embryos at
24 hpf were processed for WB. Anti-β actin antibody was used as a
loading control. (B) Quantitation of cleavage efficiency of the indicated
2As. The cleavage efficiency was calculated as depicted in Figure 2B.
P value was determined by the two-tailed Student's
t-test (n = 3). (C) Confocal
microscopy of the retina in the injected embryos at 24 hpf. Non-overlapping
green signals in the nucleus and red signals in the plasma membrane
correspond to cleaved NLS-EGFP and mCherry-CAAS, respectively. Yellow
signals in the overlay images signify uncleaved NLS-EGFP-2A-mCherry-CAAX.
The scale bar represents 20 µm.

To investigate if the outcome of human cultured cell line and zebrafish embryo
experiments is echoed in mouse tissue, we subcloned NLS-EGFP-2A-mCherry-CAAX into
the adenoviral genome via homologous recombination, transduced packaging cell lines
with the resulting recombinant adenovirus and amplified infectious adenovirus
harboring pNLS-EGFP-2A-mCherry-CAAX. Subsequently, the infectious virus particles
were injected into the tail veins of mice and livers were harvested 3 dpi. The
harvested livers were then analyzed by WB and confocal microscopy. WB analysis
displayed that P2A also has the highest cleavage efficiency in the mouse liver,
followed by T2A, E2A and F2A (Fig.
6A). Furthermore, NLS-EGFP and mCherry produced by P2A-mediated cleavage
were targeted most efficiently in the mouse liver as well, as evidenced by confocal
microscopy (Fig. 6B).
Collectively, our findings from the human cultured cell line and zebrafish embryo
experiments were reiterated in the mouse liver: P2A has the highest cleavage
efficiency, as demonstrated by WB and confocal microscopy.

Adult mice were injected with recombinant adenovirus encoding indicated
proteins via the tail vein. (A) WB analysis revealing cleavage efficiency of
the 2As in the mouse liver. The liver was extracted from the injected mice
at 3 day post-injection (dpi) and processed for WB. Anti-β actin
antibody was used as a loading control. (B) Quantitation of cleavage
efficiency of the indicated 2As. The cleavage efficiency was calculated as
depicted in Figure 2b.
The p value was determined by the two-tailed Student's
t-test (n = 3). (C) Confocal
microscopy of liver section of the injected mice. The images were taken at 3
dpi. Green, red and yellow signals are as described in the legend for Figure 5C. The scale bar
represents 20 µm.

Discussion

Here, we show that among four different 2A peptides, a 2A peptide derived from
porcine teschovirus-1 (P2A) has the highest cleavage efficiency in three human cell
lines, zebrafish embryos and mouse liver. In addition, we generated sCMV promoter
driven expression cloning vectors and adenoviral shuttle vectors, both harboring a
2A gene flanked by multiple cloning sites. These vectors can be used to
simultaneously express multiple genes in mammalian cell lines, zebrafish and mice.
Although we did not test if this is the case, it is very likely that the cloning
vectors would act in a similar manner in Xenopus, because an sCMV
promoter functions in Xenopus as well [15] and the 2A-mediated cleavage
occurs in all eukaryotic cells [9].

What is the mechanism by which P2A peptide exhibits the highest cleavage efficiency
in human cell lines, zebrafish and mouse? Ryan and colleagues proposed that there
are two ways to raise the cleavage efficiency [3]. First, insertion of a GSG
linker at the N-terminus of a 2A peptide can improve cleavage efficiency [11], [18]. Second,
addition of C-terminal amino acids of a 1D peptide to the N-terminus of a 2A peptide
can increase cleavage efficiency, as shown by the following two reports. Ryan and
colleagues reported that introduction of C-terminal five amino acids (APVKQ) of an
FMDV 1D peptide to the N-terminus of an FMDV 2A peptide increased the efficiency by
13% in vitro[9]. Groot
Bramel-Verheije and colleagues demonstrated that the introduction of C-terminal
seven amino acids (APVKQLL) of an FMDV 1D peptide to the N-terminus of an FMDV 2A
peptide raised the efficiency by 15% in both baby hamster kidney (BHK-21)
cells and porcine alveolar macrophages [19]. These two
reports, however, do not account for the difference in the cleavage efficiency among
2A peptides derived from different viruses in that the reports examined the cleavage
efficiency of the same 2A peptides in the presence or absence of extra amino acids
originating from the 1A peptide. We have demonstrated that the 19-amino acid P2A
elicits higher cleavage efficiency than the 22-amino acid 2As. This suggests that
when comparing the cleavage efficiency among 2A peptides derived from different
viruses, the length of 2A peptide is not the only factor that determines the
cleavage efficiency. Further work is required to determine the factor(s) governing
the difference in cleavage efficiency among different 2As.

We believe that identification of a highly efficient 2A peptide and construction of
expression plasmids with a 2A gene flanked by multiple cloning sites would
facilitate the use of 2A technology for simultaneous expression of multiple
genes.

Supporting Information

Two major byproducts decorated by anti-GFP antibody in lysate of cells
transfected with 2A plasmids do not appear in lysate of cells
transfected with pEGFP-N1. HEK293T cells were individually
transfected with the indicated 2A plasmids and pEGFP-N1 that does not harbor
2A sequences. The transfected cells were processed for WB 24 hr
post-transfection. The cleavage efficiency was assessed using anti-GFP
antibodies to decorate NLS-EGFP. Asterisks indicate the two major
byproducts. Note that the byproducts do not appear in lysates of cells
transfected with pEGFP-N1 (GFP). Anti-β actin antibody was used as a
loading control.

Acknowledgments

We thank Chang-Yeol Yeo for providing the pCS4+ plasmid, Sheng Nan Jiang for
technical assistance, Michael Frohman, Hyunju Ro and Hueng-Sik Choi for critical
reading of the manuscript, Geong Duck Park for support and Eun Young Choi for
encouragement.